CN105907847A - Application of primer group and method of performing genetic diversity analysis on cotton germplasm resources with the primer group - Google Patents
Application of primer group and method of performing genetic diversity analysis on cotton germplasm resources with the primer group Download PDFInfo
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Abstract
The invention relates to the technical field of molecular markers and especially relates to an application of a primer group and a method of performing genetic diversity analysis on cotton germplasm resources with the primer group. In the invention, a method of identifying Asian cotton germplasm resources with an SSR primer is disclosed, wherein the primer group includes 62 pairs of SSR core primers on 13 chromosomes in the Asian cotton A genome. These primers have basically same annealing temperature, are uniformly distributed on the 13 chromosomes, have clear bands and have good amplification effects. The method can effectively distinguish differences among different Asian cotton germplasms, is used for performing molecular-level genetic diversity analysis to the Asian cotton, provides evidence for constructing a core collection, can be used for effectively developing and utilizing high-quality germplasm resources and has important significance of cotton breeding in our country.
Description
Technical field
The present invention relates to molecular marking technique field, particularly to primer sets application, utilize this primer sets to carry out Cotton Gossypii
The method that Genetic Diversity of Germplasm is analyzed.
Background technology
Molecular marker is the genetic marker by between individuality based on hereditary material inner nucleotide sequence variations, is DNA level
The direct reflection of genetic polymorphism, can be stable hereditary, can reflect individuality and the population characteristic of biology.With other several heredity marks
Note morphology labelling, biochemical biomarker, cytological marker are compared, and DNA molecular marker has the advantage, that great majority point
Son is labeled as codominance, and the selection to recessive character is very convenient；Genome mutation is the abundantest, and the quantity of molecular marker is almost
It is unlimited；In the different phase of biological development, the DNA of different tissues can be used in labeled analysis；Molecular marker disclose from
The variation of DNA；Show as neutrality, do not affect the expression of objective trait, with bad character without chain；Detection means is simple, rapidly.
Molecular marker is the effective tool of detection Genetic Diversity of Germplasm, and molecular marking technique can not only detect the something lost in species
Pass multiformity, and the genetic diversity between species can be compared.Detect hence with molecular marker, select
Have external source objective trait gene, as far as possible without or less with unfavorable gene and heredity reach balance new germ plasm, to the modern times make
Thing improvement will be highly important.
SSR (Simple sequence repeat is called for short SSR) is also microsatellite DNA, on the basis of being built upon PCR
Second filial generation molecular marker.The polymorphism of SSR marker depends on the variation of elementary cell number of repetition, and this variation is being given birth to
It thing colony is a large amount of existence.Compared with other labelling, SSR marker quantity is enriched, and covers whole genome, announcement polymorphic
Property high；There is the characteristic of multiple alleles, it is provided that contain much information, easy and simple to handle, reproducible, be therefore widely used to,
The aspects such as genomic mapping, gene mapping, the discriminating of species sibship, gene bank structure, gene clone.SSR is at cotton gene
Distribution in group is quite varied.SSR molecular marker technology has boundless answering in the qualification of germplasm resource for cotton
Use prospect.
Asiatic cotton (G.arboreum L.) originates in the Indian subcontinent, cultivates the earliest in Asia due to it and propagates, therefore claims Asia
Continent is cotton.Asiatic cotton is long at China's cultivation history, is distributed wide, through nature and artificial selection, has defined and has been with Chinese characteristics
Germline and type, also referred to as in cotton, its germ plasm resource is rich and varied.China's Asiatic cotton is at China and World cotton gene bank
In occupy critical role.Asiatic cotton has precocity, Resistant, bell barren-resistant, rotten is few, stable yield, fiber strength are high, elastic
By force, the advantage such as cultivation management is relatively simple, therefore there are the bigger potentiality directly utilized.Effectively discover and use Asiatic cotton excellent
Germ plasm resource, significant to Cotton in China breeding
At present, having included 78,340 pairs of Cotton Gossypii SSR primers in genecotton, these primers have polymorphism height, repeat
Property good, time-consuming less and low cost and other advantages, can be as the preferred primer of Idioplasm identification.The present invention utilize 197 parts representative
Asiatic cotton Germplasms, from genecotton data base, filtered out 62 pairs of bands clear, polymorphism is high, amplification effect
The best SSR primer, it is intended to carry out the genetic diversity Journal of Sex Research of molecular level for Asiatic cotton, Core Germplasms builds, for effectively sending out
Pick utilizes Asiatic cotton quality germplasm to provide foundation.
Summary of the invention
In view of this, the present invention provides the application of primer sets, utilizes this primer sets to carry out germplasm resource for cotton genetic diversity
Property analyze method.It is an object of the invention to provide one group and be suitable for Asiatic cotton germplasm identification, analysis of genetic diversity
Core primers and test method, and provide convenient for similar research.
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
The invention provides the primer sets selected from having nucleotide sequence as follows to identify in Germplasm Resources of Upland Cotton
In application；
Present invention also offers the application in germplasm resource for cotton analysis of genetic diversity of the described primer sets.
A kind of method that present invention also offers germplasm resource for cotton analysis of genetic diversity, comprises the steps:
Step 1: extract the STb gene obtaining sample；
Step 2: with step 1 obtain STb gene as template, expand with primer sets as claimed in claim 1, electrophoresis
Detection, colour developing, carry out polymorphism statistical analysis.
In some specific embodiments of the present invention, described polymorphism statistical analysis is by 0, and 1 adds up, and uses
Genetic similarity between Jaccard coefficient calculations sibling species, utilizes unweighted mean UPGMA method on the basis of genetic similarity
All materials to be tested are set up dendrogram, similarity coefficient and cluster analysis Powe marker V3.25 software complete.
In some specific embodiments of the present invention, described in step 2,10 μ L reaction systems of amplification include 10 × PCR
(containing 20mM Mg2+) 1 μ L, dNTP (10mM) 0.2 μ L, Taq enzyme (2.5U/ μ L) 0.2 μ L, deionization sterilized water 6.1 μ L, upstream is drawn
Thing (5 μMs) 0.75 μ L, downstream primer (5 μMs) 0.75Powe marker V3.25, template DNA (50ng/ μ L) 1 μ L.
In some specific embodiments of the present invention, described in step 2, the response procedures of amplification is 95 DEG C of degeneration 2min；
94 DEG C of degeneration 40s, 57 DEG C of annealing 45s, 72 DEG C extend 60s, totally 30 circulations；72 DEG C extend 7min.
In some specific embodiments of the present invention, it is extracted as described in step 1: take cotton leaf needle a piece of, put
Enter in 2mL centrifuge tube, be simultaneously charged into a steel ball, be placed in liquid nitrogen freezing 0.5h；Take out centrifuge tube and put into grinding 2 in beveller
Secondary, each 30S；Take out steel ball after grinding, add the extracting solution of 60 DEG C of water-bath preheatings of 700 μ L；60 DEG C of water-bath 40-60min, often
10min shakes centrifuge tube；Take out centrifuge tube, add the volume ratio of isopyknic chloroform-isoamyl alcohol mixture, chloroform and isoamyl alcohol
For 24:1, mixing；12,000g, centrifugal 10min；Take supernatant, be placed in 2ml centrifuge tube, add the mixing of isopyknic chloroform-isoamyl alcohol
Thing, chloroform is 24:1 with the volume ratio of isoamyl alcohol；Mixing；12,000g, centrifugal 10min；Take supernatant, be placed in 2ml centrifuge tube, add
The isopropanol of 0.6 times of volume；The most reverse centrifuge tube, until there being flocculent deposit, stands 10min；Go supernatant or hook to go out DNA to arrive
In the centrifuge tube of 1.5mL, washing 2～3 times with 70% ethanol, dehydrated alcohol is washed once, vacuum drying；Add 500uL TE dissolving DNA,
It is stored in-20 DEG C of refrigerators standby.
In some specific embodiments of the present invention, described electrophoresis detection is: the polyacrylamide gel of 8%, specifically
For: preparation gel, acrylamide: methylene diacrylamide (Acr:Bis30) 6.7mL, 5 × TBE 5mL, deionized water (dH2O)
13.25mL, 10%AP (10% Ammonium persulfate. 350 μ L, tetramethylethylenediamine (TEMED) 11 μ L；Electrophoresis 1h.
In some specific embodiments of the present invention, described colour developing is that the glue after power taking swimming is placed on acetic acid and dehydrated alcohol
And fix 7-8min in the fixative of pure water preparation, permeate 10min with silver nitrate penetrating fluid；It is placed on containing hydrogen-oxygen after washing twice
Change and develop the color in the nitrite ion of sodium, formaldehyde, until brown band occurs in glue, be placed on and terminate containing in Carbon Dioxide sodium solution.
Present invention have the advantage that:
The invention discloses 62 pairs of Cotton Gossypii core SSR primers, be distributed evenly on 13 chromosomes, these primer polymorphisms
Good, band is clear, is used directly for the qualification of Asiatic cotton variety source, and analysis of genetic diversity eliminates common experiment
During the screening process of SSR primer, improve conventional efficient.Disclosed by the invention the 62 pairs of SSR core primers are utilized to identify Asia
The method of continent cotton germ plasm resource, experimental implementation is simple, and result is good, can effectively distinguish the Asia cotton seed matter money of separate sources background
Source material, is the method for the germplasm identification of a kind of worth high praise.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
In having technology to describe, the required accompanying drawing used is briefly described.
Fig. 1 shows that 24 represent the material the selection result to primer NAU5233 and NAU5351；Wherein, 1-24 is material respectively
Material: the Guichi white seed of little unginned cotton, cotton in Fengyang, Guichi, Anhui crow seed, cotton in the richness clock of Guangxi, cotton in the Zuo Xian of Guangxi, Anlong little Hua, Wan County
Purple straw edelweiss, sub-No. 1 of stone system, Shangqiu edelweiss, cotton in the purple stalk of Central China, Changshu, Jiangsu Province dragon species 1, edelweiss, storehouse, Siyang collection is blue or green
Cotton in stem, the white seed in granary (clear), cotton in Flos Nelumbinis, hophornbeam is red, Ramulus et Folium Bischofiae Javanicae black race, the little cotton of Jiyang Area, Shandong, cotton in Jinghong, Pinghu microspecies ash
Seed, cotton in brown son, cotton in Pingguo dawn, middle print hybrid cotton 4, the black seed of Bai Mila Hemerocallis citrina Baroni；
Fig. 2 shows the primer NAU2026 amplification figure to 197 material 1～No. 48 materials therein；1-48 swimming lane is the most right
Answer 1-48 material (title material sees attached list 3, the same Fig. 1 of Marker)；
Fig. 3 shows the amplification figure being primer NAU2026 to 197 material 49～No. 96 materials therein；1-48 swimming lane divides
Not corresponding 49-96 material (title material sees attached list 3, the same Fig. 1 of Marker)；
Fig. 4 shows the primer NAU2026 amplification figure to 197 material 97～No. 144 materials therein；1-48 swimming lane is respectively
Corresponding 97-144 material (title material sees attached list 3, the same Fig. 1 of Marker)
Fig. 5 shows the primer NAU2026 amplification figure to 197 material 145～No. 192 materials therein；1-48 swimming lane divides
Not corresponding 145-192 material (title material sees attached list 3, the same Fig. 1 of Marker)；
Fig. 6 shows the primer NAU2026 amplification figure to 197 material 193～No. 197 materials therein；1-5 swimming lane divides
Not corresponding 193-197 material (title material is shown in Table 3, the same Fig. 1 of Marker)；
Fig. 7 shows the sibship dendrogram of 197 parts of Asiatic cotton materials；
Fig. 8 shows without polymorphism primer MON_CGR5118 and MON_CGR 5127 amplification of 24 sieve primer materials in table 2
Design sketch；
Fig. 9 shows in table 2 24 materials screening primers, amplification without the bad primer MON_CGR5426 of polymorphism and effect with
MON_CGR5429 schematic diagram.
Detailed description of the invention
The invention discloses the application of primer sets, utilize this primer sets to carry out germplasm resource for cotton analysis of genetic diversity
Method, those skilled in the art can use for reference present disclosure, is suitably modified technological parameter and realizes.Special needs to be pointed out is, institute
Having similar replacement and change apparent to those skilled in the art, they are considered as being included in the present invention.
Method and the application of the present invention are described by preferred embodiment, and related personnel substantially can be without departing from the present invention
In appearance, spirit and scope, method described herein and application it is modified or suitably changes and combine, realizing and apply this
Inventive technique.
The invention provides the core primers of Asiatic cotton germplasm identification.
62 pairs of described its sequences of SSR primer and chromosome position information are shown in Table 1.
Present invention also offers the application in germplasm resource for cotton is identified of the described 62 pair SSR primer
62 pairs of described SSR primers, in the method for germplasm resource for cotton analysis of genetic diversity, comprise the steps:
(1) DNA extraction
Utilize the CTAB method of improvement, extract the STb gene of sample.
(2) primer amplification and electrophoresis
The DNA profiling extracted with step (1), the primer described in utilization carries out PCR amplification, the PCR primer of step (2) is entered
Row agarose gel electrophoresis detects, and then silver staining colour developing, finally carries out polymorphism statistical analysis.
(3) data are added up by 0,1, with genetic similarity between Jaccard coefficient calculations sibling species, at genetic resemblance
Utilize unweighted mean UPGMA method that all materials to be tested are set up dendrogram, similarity coefficient and cluster analysis on coefficient basis to use
Powe marker V3.25 software completes.
(4) primer described in be from genecotton data base (Http:// genecotton.com/), in, according to primer
The position of chromosome, pick out, then with 24 representational screening varieties in 197 parts of Asiatic cotton Germplasms
Out.
SSR primer carries out the method for germplasm resource for cotton qualification, it is characterized in that described method comprises the steps:
The extraction of DNA:
1. take cotton leaf needle a piece of, put in 2mL centrifuge tube, be simultaneously charged into a steel ball, be placed in liquid nitrogen freezing
Half an hour；
2. take out centrifuge tube with strainer and put into grinding 2 times, each 30S in beveller；
3. take out steel ball after grinding and add 700ul extracting solution (60 DEG C of water-bath preheatings)；
4.60 DEG C of water-baths 40-60 minute (during this, every 10 minutes shake centrifuge tubes)；
5. take out centrifuge tube, add equal-volume chloroform: isoamyl alcohol (24:1), slowly mixing (overturns centrifuge tube about 50 times)；
6. put into centrifuge, 12,000g, centrifugal 10 minutes；
7. take supernatant, be placed in 2ml centrifuge tube, add equal-volume chloroform: isoamyl alcohol (24:1).Repeat 5,6 steps；
8. take supernatant, be placed in 2ml centrifuge tube, add the ice-cold isopropanol of 0.6 times of volume；
9. the most reverse centrifuge tube, until there being flocculent deposit, stands 10 minutes.
10. removing supernatant (or hook goes out in the centrifuge tube of DNA to 1.5ml), wash 2-3 time with 70% ethanol, dehydrated alcohol is washed
Once, vacuum drying
11. add 500uL TE dissolving DNA, are stored in-20 DEG C of refrigerators standby.
10 μ l PCR reaction system: 10 × PCR (containing 20mMMg2+) 1 μ l, dNTP (10mM) 0.2 μ l of described step (2),
Taq enzyme (2.5U/ μ l) 0.2 μ l, deionization sterilized water 6.1 μ l, F.P. (5 μMs) 0.75 μ l, R.P. (5 μMs) 0.75 μ l, template DNA
(50ng/μl)1μl.
The PCR response procedures of described step (2): 95 DEG C of degeneration 2 minutes；94 DEG C of degeneration 40 seconds, anneal 45 seconds for 57 DEG C, 72 DEG C
Extend 60 seconds, totally 30 circulations；72 DEG C extend 7 minutes.
The electrophoresis detection of described step (2) is: the polyacrylamide gel of 8%.Particular order is: preparation gel, propylene
Amide: methylene diacrylamide (Acr:Bis30) 6.7ml, 5 × TBE 5ml, deionized water (dH2O) 13.25ml, 10%AP
(10% Ammonium persulfate. 350 μ l, tetramethylethylenediamine (TEMED) 11 μ l.Install electrophoresis tank glass, encapsulating, after gelling is solid,
Pull up comb, encapsulating, electrophoresis 1 hour.
The dyeing course of described step (2): 1: be placed in the fixative of acetic acid and dehydrated alcohol and pure water preparation and fix 7-8
Minute, then permeate 10 minutes with silver nitrate penetrating fluid；Pure water is placed on containing sodium hydroxide, the nitrite ion of formaldehyde after washing twice
In develop the color, until brown band occurs in glue, be finally placed on and terminate containing in Carbon Dioxide sodium solution.
The glue contaminated is placed on film viewer photograph, by there being band to be designated as 1, is designated as 0 without band and adds up.
Present invention have the advantage that:
The invention discloses 62 pairs of Cotton Gossypii core SSR primers, be distributed evenly on 13 chromosomes, these primer polymorphisms
Good, band is clear, is used directly for the qualification of Asiatic cotton variety source, and analysis of genetic diversity eliminates common experiment
During the screening process of SSR primer, improve conventional efficient.
The method utilizing 62 pairs of SSR core primers to identify Asiatic cotton germ plasm resource disclosed by the invention, experimental implementation is simple,
Result is good, can effectively distinguish the Asiatic cotton Germplasms of separate sources background, is the germ plasm resource of a kind of worth high praise
The method identified.
The present invention provide primer sets application, utilize this primer sets to carry out germplasm resource for cotton analysis of genetic diversity
In method, raw materials used and reagent all can be buied by market.
Below in conjunction with embodiment, the present invention it is expanded on further:
The analysis of genetic diversity of 1 197 parts of Asiatic cotton germ plasm resources of embodiment, specifically comprises the following steps that
197 parts of Asia cotton seed matter are selected according to cultivar origin and phenotypic characteristic from National Cotton kind matter storehouse in mid-term, as
Experiment material.
7500 pairs of SSR primers of this laboratory are screened with 24 representational materials.Finally screen 62 pairs of bands clear,
It is prone to the SSR primer of the polymorphism of statistics.
With above-mentioned 62 pairs of primers, to above-mentioned 197 parts of Asia cotton seed matter, expand, electrophoresis silver staining.
The PCR response procedures of amplification: 95 DEG C of degeneration 2 minutes；94 DEG C of degeneration 40 seconds, anneal 45 seconds for 57 DEG C, and 72 DEG C extend 60
Second, totally 30 circulations；72 DEG C extend 7 minutes.
The method of electrophoresis:
The polyacrylamide gel of 8%.Particular order is: preparation gel, acrylamide: methylene diacrylamide (Acr:
Bis30) 6.7ml, 5 × TBE 5ml, deionized water (dH2O) 13.25ml, 10%AP (10% Ammonium persulfate. 350 μ l, tetramethyl
Ethylenediamine (TEMED) 11 μ l.Install electrophoresis tank glass, encapsulating, treat glue.After solidification, pulling up comb, encapsulating, electrophoresis 1 is little
Time.
Dyeing course: 1: be placed in the fixative of acetic acid and dehydrated alcohol and pure water preparation and fix 7-8 minute, then use nitre
Acid silver penetrating fluid permeates 10 minutes；Pure water wash be placed on after twice containing sodium hydroxide, formaldehyde nitrite ion in develop the color, until glue
Till brown band occurs, finally it is placed on and terminates containing in Carbon Dioxide sodium solution.
Data statistics: data are added up by 0,1, with genetic similarity between Jaccard coefficient calculations sibling species, is losing
Utilize unweighted mean UPGMA method that all materials to be tested are set up dendrogram, similarity coefficient and cluster on the basis of passing similarity coefficient
Analysis Powe marker V3.25 software completes.
Result is as follows:
Utilize PopGen3.2 that the amplification of 62 pairs of primers is analyzed, the 62 SSR primers to having pleomorphism site
In 197 parts of Asiatic cotton materials, coamplification goes out 218 allele, and wherein polymorphic allele number 178, account for 81.7%.
Every pair of primer allele luffing is 2～5, average out to 2.6, and the excursion of loci polymorphism quantity of information (PIC) is
0.17～0.79, Shannon PIC value average out to 0.633.62 pairs of SSR primers selected by explanation have in 197 parts of Asiatic cottons
There is the polymorphism of higher level.
Use Powermarker V 3.25 that 197 Upland Cottons are carried out UPGMA cluster analysis, (see Fig. 7) result,
When threshold value is 0.17,197 parts of Asiatic cottons can gather is 3 big classes.The first kind includes 78 parts of Asiatic cotton materials, and the first kind can be entered
One step is subdivided into two subclass, includes 37 and 41 parts of Asiatic cotton materials respectively；Equations of The Second Kind includes 77 parts of Asiatic cotton materials, the
Two classes can be further subdivided into three subclass, includes 26,33 and 18 parts of Asiatic cotton materials respectively；3rd class includes 42 parts
Asiatic cotton material.In terms of cluster result, SSR monoid and geographic origin the most necessarily relation, each monoid source is the most identical,
Have the most more disperses, this extensively introducing a fine variety and propagating relevant with Asiatic cotton, also illustrates that each cotton region multiformity is the abundantest；But
From the point of view of between little monoid or monoid, the kind matter in same cotton region of originating still tends to getting together, and illustrates that geographic origin is close
Some Species matter sibship is nearer.Research also finds, part has the kind matter of some very special quality character and clusters based on SSR marker
Time also gather together, illustrate that qualitative trait more can reflect the hereditary variation between kind of matter.
In a word, the SSR primer of these 62 pairs of polymorphisms that testing sieve is selected can have more much higher sample, and can be this reality
Test used 197 part Asiatic cotton to classify, the reflected well genetic diversity of these Asiatic cotton materials.
1 62 pairs of primer information of table
The representative material of 2 24, table screening primer
197 parts of Asiatic cotton materials used by table 3 application example
Matched group
Select 24 parts of Asiatic cotton materials, to without polymorphism primer MON_CGR5118 and MON_CGR5127, MON_CGR5426
Screening with MON_CGR5429, method is with embodiment 1.
Result is shown in Fig. 8, Fig. 9.
Visible, the SSR primer of 62 pairs of polymorphisms that the present invention provides can have more much higher sample, and can be this experiment
197 parts of used Asiatic cottons classify, the reflected well genetic diversity of these Asiatic cotton materials.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For Yuan, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (9)
1. it is selected from the application in Germplasm Resources of Upland Cotton is identified of the primer sets with nucleotide sequence as follows；
The primer sets the most according to claim 1 application in germplasm resource for cotton analysis of genetic diversity.
3. the method for a germplasm resource for cotton analysis of genetic diversity, it is characterised in that comprise the steps:
Step 1: extract the STb gene obtaining sample；
Step 2: with step 1 obtain STb gene as template, expand with primer sets as claimed in claim 1, electrophoresis examine
Survey, colour developing, carry out polymorphism statistical analysis.
Method the most according to claim 3, it is characterised in that described polymorphism statistical analysis is by 0, and 1 adds up, uses
Genetic similarity between Jaccard coefficient calculations sibling species, utilizes unweighted mean UPGMA method on the basis of genetic similarity
All materials to be tested are set up dendrogram, similarity coefficient and cluster analysis Powe marker V3.25 software complete.
5. according to the method described in claim 3 or 4, it is characterised in that described in step 2,10 μ L reaction systems of amplification include
10 × PCR is (containing 20mM Mg2+) 1 μ L, dNTP (10mM) 0.2 μ L, Taq enzyme (2.5U/ μ L) 0.2 μ L, deionization sterilized water 6.1 μ L,
Forward primer (5 μMs) 0.75 μ L, downstream primer (5 μMs) 0.75Powe marker V3.25, template DNA (50ng/ μ L) 1 μ L.
6. according to the method described in any one of claim 3 to 5, it is characterised in that described in step 2, the response procedures of amplification is
95 DEG C of degeneration 2min；94 DEG C of degeneration 40s, 57 DEG C of annealing 45s, 72 DEG C extend 60s, totally 30 circulations；72 DEG C extend 7min.
7. according to the method described in any one of claim 3 to 6, it is characterised in that be extracted as described in step 1: take cotton leaf
Sheet needle is a piece of, puts in 2mL centrifuge tube, is simultaneously charged into a steel ball, is placed in liquid nitrogen freezing 0.5h；Taking-up centrifuge tube is put into
Beveller grinds 2 times, each 30S；Take out steel ball after grinding, add the extracting solution of 60 DEG C of water-bath preheatings of 700 μ L；60 DEG C of water-baths
40-60min, every 10min shake centrifuge tube；Taking out centrifuge tube, add isopyknic chloroform-isoamyl alcohol mixture, chloroform is with different
The volume ratio of amylalcohol is 24:1, mixing；12,000g, centrifugal 10min；Take supernatant, be placed in 2ml centrifuge tube, add isopyknic chlorine
Imitative-iso pentane alcohol mixture, chloroform is 24:1 with the volume ratio of isoamyl alcohol；Mixing；12,000g, centrifugal 10min；Take supernatant, be placed in
2ml centrifuge tube, adds the isopropanol of 0.6 times of volume；The most reverse centrifuge tube, until there being flocculent deposit, stands 10min；Go
Clear or hook goes out in the centrifuge tube of DNA to 1.5mL, washes 2～3 times with 70% ethanol, and dehydrated alcohol is washed once, vacuum drying；Add
500uL TE dissolving DNA, is stored in-20 DEG C of refrigerators standby.
8. according to the method described in any one of claim 3 to 7, it is characterised in that described electrophoresis detection is: the polypropylene of 8%
Acrylamide gel, particularly as follows: preparation gel, acrylamide: methylene diacrylamide (Acr:Bis30) 6.7mL, 5 × TBE 5mL,
Deionized water (dH2O) 13.25mL, 10%AP (10% Ammonium persulfate. 350 μ L, tetramethylethylenediamine (TEMED) 11 μ L；Electrophoresis
1h。
9. according to the method described in any one of claim 3 to 8, it is characterised in that described colour developing is that the glue after power taking swimming is placed on
Fix 7-8min in the fixative of acetic acid and dehydrated alcohol and pure water preparation, permeate 10min with silver nitrate penetrating fluid；Wash twice
After be placed on containing sodium hydroxide, formaldehyde nitrite ion in develop the color, until brown band occurs in glue, be placed on containing natrium carbonicum calcinatum
Solution terminates.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610246532.2A CN105907847B (en) | 2016-04-20 | 2016-04-20 | The application of primer sets, the method for carrying out germplasm resource for cotton analysis of genetic diversity using the primer sets |
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| CN106967833A (en) * | 2017-05-22 | 2017-07-21 | 中国农业科学院棉花研究所 | The primer and its PCR authentication methods identified for diploid A genomes cotton seed and/or tetraploid cotton seed |
| CN107190053A (en) * | 2017-03-13 | 2017-09-22 | 北京林业大学 | The combination of cypress microsatellite molecular marker, primer screening method and its application |
| CN109762923A (en) * | 2019-03-07 | 2019-05-17 | 中国农业科学院棉花研究所 | With the SSR molecular marker of upland cotton breeding time and plant villus close linkage |
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| CN107190053A (en) * | 2017-03-13 | 2017-09-22 | 北京林业大学 | The combination of cypress microsatellite molecular marker, primer screening method and its application |
| CN106967833A (en) * | 2017-05-22 | 2017-07-21 | 中国农业科学院棉花研究所 | The primer and its PCR authentication methods identified for diploid A genomes cotton seed and/or tetraploid cotton seed |
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| CN109762923A (en) * | 2019-03-07 | 2019-05-17 | 中国农业科学院棉花研究所 | With the SSR molecular marker of upland cotton breeding time and plant villus close linkage |
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| CN105907847B (en) | 2018-09-25 |
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